Patent application title: Method and device for the multiplex analysis of cells and tissues

Abstract:

This invention is used for the multiplex analysis method of cells or
tissues by combination of multiple cubicles and permeable membrane.
Utilizing the diffusion based reagents administration; cells and tissues
in multiple cubicles receive signals evenly in their cubicles as parallel
manner. This device makes efficient multiplex cells or tissues assays
with simple plate layout. According to the molecular permeable mechanism,
single spike of stimuli as chemical trigger, light activation, electric
triggers or temperature shift can induce multiple cells or tissues
response at once. Then accompanying with calibrator molecules as for the
navigation of stimuli and assisted by suitable computer simulation,
accurate kinetic cells or tissues response analysis is achieved.

Claims:

1. Method of multiplex cells or tissue analysis with the culture vessel of
separate compartment cubicles and permeable materials.

2. The method of multiplex cells or tissue analysis of claim 1, wherein
said method is Kinetic analysis method by evoked with diffusing
chemicals.

7. The method of multiplex cells or tissue analysis of claim 1, wherein
said method is accompany with the navigation molecules as colored dyes or
fluorescent dyes as kinetic calibrator.

8. The method of multiplex cells or tissue analysis of claim 1, wherein
said the data of kinetic analysis is compensated by computer simulation
technique.

Description:

BACKGROUND OF THE INVENTION

[0001]The cell culture technique has been used in many biological research
fields and it expands their utilities. Different cells, tissues, culture
medium and culture wears are use in order to increase the efficiency of
output. According to the necessity of assays volume, multiple well plates
were incorporated in past 30 years.

[0002]Then, according to the achievement of more native cell or tissue
environment, 3D cell culture technique is also getting popular in cells
or tissue cultures. Special culture wares were invented for the efficient
3D cell culturing technique too. (Ref 1-5)

[0003]By the complexity of 3D culture, the multiple processing and image
acquisition of cells or tissues are quite difficult. In this patent
application, we apply the permeable membrane with separate cubicles for
cell culture, especially 3D cell culture for increasing output by
incorporating multiplexing assays. Then this patent application shows a
successful approach to establish the efficient multiplex assay method for
cells or tissue analysis.

SUMMARY OF THE INVENTION

[0004]At first, multiple cubicles are formed in the bottom of microplate
or culture insert. Then permeable membranes are installed in order to
separate these cubicles. Such permeable membranes can form the
extra-space for the reagent additional part between these cubicles too.
This extra-space is used for the chemical administration and
drug-releasing portion. Cells or tissues exist in each cubicle and they
are observed by conventional optical modality as well as more 3D
adaptable optics. Multi-photon observation is preferable. Additional
calibration molecules as colored dyes or fluorescence molecules assist
more accurate observation. Then, computer assisted simulation will
compensated the data and increase the quality of results.

EMBODIMENT 1

Multiplex Analysis of Cells or Tissue in Single Reaction Vessel (FIG. 1).

[0005]Multiple cubicles (1) are formed in the bottom of culture well or
culture insert (2). The permeable membrane (3) separates each cubicle
(1). Cells or tissues (C) are located in each cubicles (1) relatively
stable manner. If the culture medium contains gelling materials, these
cells or tissues are more stable in each cubicle (1). The bottom (2) of
the cubicles is transparent for the optical measurement from bottom side.
After the administration of reagent (4) on the top layer of cubicles (1),
small molecules (S) permeate into the cubicles by time dependent manner.
And all cells or tissues (C) receive stimulus uniformly. According to the
simple experiments, the diffusion time needs about 30 minutes crossing
the 8 mm traveling when the medium contains 0.5% gelatin in PBS/0.1%
Tween 20 at room temperature (FIG. 2). The kinetics of diffusion speed
varies by their environment as temperature, ionic concentration,
culturing materials and molecular weight of substances. So the
measurement is compensated by calibrator dyes and computer simulation.

EMBODIMENT 2

Multiple Analysis of Cells or Tissue with Extra Reagent Space in Single
Reaction Vessel (FIG. 3)

[0006]Multiple cubicles (1) are formed in the bottom of culture well or
culture insert (2). The permeable membrane (3) separates each cubicle (1)
and prepares extra space (5). Cells or tissues (C) are located in each
cubicle (1). The bottom (2) of the cubicles is transparent for the
optical measurement from. Reagent (6) is administrated into the extra
space (5). Small molecules (S) permeate into the all cubicles by time
dependent manner and cells or tissues (C) receive stimuli evenly. The
kinetics of diffusion speed varies by their environment as temperature,
ionic concentration, culturing materials and molecular weight of
substances. So the measurement is compensated by calibrator dyes and
computer simulation.